Arrays of integrated analytical devices and methods for production

ABSTRACT

Arrays of integrated analytical devices and their methods for production are provided. The arrays are useful in the analysis of highly multiplexed optical reactions in large numbers at high densities, including biochemical reactions, such as nucleic acid sequencing reactions. The integrated devices allow the highly sensitive discrimination of optical signals using features such as spectra, amplitude, and time resolution, or combinations thereof. The arrays and methods of the invention make use of silicon chip fabrication and manufacturing techniques developed for the electronics industry and highly suited for miniaturization and high throughput.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/600,668, filed on May 19, 2017, which is a continuation of U.S.patent application Ser. No. 15/158,756, filed on May 19, 2016, now U.S.Pat. No. 9,658,161, which is a continuation of U.S. patent applicationSer. No. 13/920,037, filed on Jun. 17, 2013, now U.S. Pat. No.9,372,308, which claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 61/660,776, filed on Jun. 17, 2012, thedisclosures of which are incorporated herein by reference in theirentireties.

BACKGROUND OF THE INVENTION

In analytical systems, the ability to increase the number of analysesbeing carried out at any given time by a given system has been a keycomponent to increasing the utility and extending the lifespan of suchsystems. In particular, by increasing the multiplex factor of analyseswith a given system, one can increase the overall throughput of thesystem, thereby increasing its usefulness while decreasing the costsassociated with that use.

In optical analyses, increasing multiplex often poses increaseddifficulties, as it may require more complex optical systems, increasedillumination or detection capabilities, and new reaction containmentstrategies. In some cases, systems seek to increase multiplex by manyfold, and even orders of magnitude, which further implicate theseconsiderations. Likewise, in certain cases, the analytical environmentfor which the systems are to be used is so highly sensitive thatvariations among different analyses in a given system may not betolerable. These goals are often at odds with a brute force approach ofsimply making systems bigger and of higher power, as such steps oftengive rise to even greater consequences, e.g., in inter reactioncross-talk, decreased signal to noise ratios resulting from either orboth of lower signal and higher noise, and the like. It would thereforebe desirable to provide analytical systems that have substantiallyincreased multiplex for their desired analysis, and particularly for usein highly sensitive reaction analyses, and in many cases, to do so whileminimizing negative impacts of such increased multiplex.

At the same time, there is a continuing need to increase the performanceof analytical systems and reduce the cost associated with manufacturingand using the system. In particular, there is a continuing need toincrease the throughput of analytical systems. There is a continuingneed to reduce the size and complexity of analytical systems. There is acontinuing need for analytical systems that have flexible configurationsand are easily scalable.

SUMMARY OF THE INVENTION

The instant invention addresses these and other problems by providing inone aspect arrays of integrated analytical devices comprising:

a substrate layer;

a filter module layer disposed on the substrate layer;

a collection module layer disposed on or with the filter module layer;

a waveguide module layer disposed on the collection module layer;

a zero-mode waveguide module layer disposed on the waveguide modulelayer;

wherein the zero-mode waveguide module layer comprises a plurality ofnanometer-scale apertures penetrating into the waveguide module layer.

In some embodiments, the substrate layer is a detector layer.

In specific embodiments, the substrate layer is a CMOS wafer detectorlayer.

In some embodiments, the filter module layer comprises a dielectricfilter.

In other embodiments, the filter module layer comprises an absorptivefilter.

In specific embodiments, the detector layer comprises a color-separationlayer.

According to some embodiments, the plurality of nanometer-scaleapertures is formed by etching, and the etching is stopped using anendpoint signal.

In specific embodiments, the waveguide module layer comprises an uppercladding of low n material disposed on a high n material, and at leastone nanometer-scale aperture fully penetrates the upper cladding of lown material into the high n material. In more specific embodiments, theat least one nanometer-scale aperture is partially backfilled. In evenmore specific embodiments, the at least one nanometer-scale aperture ispartially backfilled using atomic layer deposition or low pressurechemical vapor deposition. In some specific embodiments, the uppercladding of low n material is SiO₂, and in some specific embodiments,the high n material is Si₃N₄. In some specific embodiments, the arraysof integrated analytical devices further comprise an etch hardmaskdisposed between the high n material and the upper cladding of low nmaterial.

In some embodiments, the collection module layer of the instant arraysof integrated analytical devices comprises a Fresnel lens structure. Inspecific embodiments, the Fresnel lens structure is a phase Fresnel zoneplate.

In preferred embodiments of the instant arrays, at least onenanometer-scale aperture comprises a fluid sample that comprises afluorescent species. In even more preferred embodiments, the fluorescentspecies is a fluorescently labeled nucleotide analog.

In specific embodiments, the plurality of nanometer-scale aperturescomprise at least 100 nanometer-scale apertures. In other specificembodiments, the plurality of nanometer-scale apertures have a densityof at least 1000 apertures per cm².

In another aspect, the invention provides methods for producing an arrayof integrated analytical devices comprising:

providing a substrate layer;

depositing a filter module layer on the substrate layer;

depositing a collection module layer on the filter module layer;

patterning and etching the filter module layer and the collection modulelayer to form an array of protrusions having tops and sides and havinggaps between the protrusions;

depositing a reflective material on the array of protrusions such thatthe tops and sides of the protrusions comprise a reflective layer;

depositing a fill material on the reflective layer such that the fillmaterial fills the gaps between the protrusions;

patterning and etching the fill material and reflective layer to removethe reflective layer from the tops of the protrusions;

depositing a first layer of low n material on the etched fill materialand the tops of the protrusions;

depositing a high n material on the first layer of low n material;

depositing a second layer of low n material on the high n material toform an upper cladding and to complete a waveguide module layer disposedon the collection module layer;

depositing a zero-mode waveguide material on the surface of thewaveguide module layer to form a zero-mode waveguide module layer;

patterning and etching the zero-mode waveguide module layer to define aplurality of nanometer-scale apertures penetrating into the uppercladding of the waveguide module layer.

In specific embodiments, the methods further comprise the step ofpatterning and etching the high n material to define a waveguide.

In other specific embodiments, the substrate layer is a detector layer.

In more specific embodiments, the substrate layer is a CMOS wafer.

In certain embodiments, the filter module layer comprises a dielectricfilter.

In other embodiments, the filter module layer comprises an absorptivefilter.

In specific embodiments, the substrate layer comprises acolor-separation layer.

According to some embodiments, the step of etching the zero-modewaveguide module layer is stopped using an endpoint signal, and in someembodiments the zero-mode waveguide module layer is etched until atleast one nanometer-scale aperture fully penetrates the upper claddingof the waveguide module layer.

In some embodiments, the methods further comprise the step of partiallybackfilling at least one nanometer-scale aperture, where, in someembodiments, the step of partially backfilling the at least onenanometer-scale aperture uses atomic layer deposition or low pressurechemical vapor deposition.

In some embodiments, the methods further comprise the step depositing anetch hardmask on the high n material prior to forming the upper claddingand completing the waveguide module layer.

In some embodiments, the second layer of low n material is SiO₂, and insome embodiments, the high n material is Si₃N₄.

In specific embodiments, the plurality of nanometer-scale aperturescomprise at least 100 nanometer-scale apertures, and in other specificembodiments, the plurality of nanometer-scale apertures have a densityof at least 1000 apertures per Cm².

In yet another aspect, the invention provides methods for producing anarray of integrated analytical devices comprising:

providing a substrate layer;

depositing a filter module layer on the substrate layer;

depositing a collection module layer on the filter module layer, whereinthe collection module layer comprises a Fresnel lens;

depositing a first layer of low n material on the collection modulelayer;

depositing a high n material on the first layer of low n material;

depositing a second layer of low n material on the high n material toform an upper cladding and to complete a waveguide module layer;

depositing a zero-mode waveguide material on the surface of thewaveguide module layer to form a zero-mode waveguide module layer;

patterning and etching the zero-mode waveguide module layer to define aplurality of nanometer-scale apertures penetrating into the uppercladding of the waveguide module layer.

In specific embodiments, the methods comprise the step of patterning andetching the high n material to define a waveguide.

In other specific embodiments, the substrate layer is a detector layer.

In still other specific embodiments, the substrate layer is a CMOSwafer.

In some embodiments, the filter module layer comprises a dielectricfilter.

In some embodiments, the filter module layer comprises an absorptivefilter.

In specific embodiments, the detector layer comprises a color-separationlayer.

According to some embodiments, etching of the zero-mode waveguide modulelayer is stopped using an endpoint signal.

In specific embodiments, the zero-mode waveguide module layer is etcheduntil at least one nanometer-scale aperture fully penetrates the uppercladding of the waveguide module layer.

In some embodiments, the methods further comprise the step of partiallybackfilling at least one nanometer-scale aperture.

In specific embodiments, the step of partially backfilling the at leastone nanometer-scale aperture uses atomic layer deposition or lowpressure chemical vapor deposition.

In some embodiments, the methods further comprise the step of depositingan etch hardmask on the high n material prior to forming the uppercladding and completing the waveguide module layer. In some specificembodiments, the second layer of low n material is SiO₂, and in somespecific embodiments, the high n material is Si₃N₄.

In specific embodiments, the plurality of nanometer-scale aperturescomprise at least 100 nanometer-scale apertures, and in other specificembodiments, the plurality of nanometer-scale apertures have a densityof at least 1000 apertures per Cm².

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-B schematically illustrates an exemplary nucleic acid sequencingprocess that can be carried out using aspects of the invention.

FIG. 2 provides a schematic block diagram of an integrated analyticaldevice.

FIG. 3A provides a schematic of excitation spectra for two signal eventsand an indicated narrow band excitation illumination, while FIG. 3Bschematically illustrates the resulting detected signal based upon thenarrow band illumination of the two signal events.

FIG. 4 schematically illustrates the signal profiles for each of fourfluorescent labeling groups, overlain with each of two different filterprofiles.

FIG. 5 illustrates modeled signal data plotted as a function of detectedchannel 1 and channel 2 intensity.

FIG. 6 schematically illustrates an integrated analytical device fordetecting signals as shown in FIG. 5.

FIG. 7 schematically illustrates signal traces for a two-color,two-amplitude sequence-by-synthesis reaction.

FIG. 8A schematically illustrates emission spectra of four distinctsignal events, e.g., fluorescently labeled nucleotide analogs; FIG. 8Bschematically illustrates a signal profile at each detector elementbased upon a typical four-color separation scheme.

FIG. 9 schematically illustrates a similar signal profile at each offour detector elements, but based upon an alternative filterarchitecture.

FIG. 10 schematically illustrates an exemplary array of integratedanalytical devices, where each device comprises a dielectric filterlayer within the reflective cone, and there is no color separation inthe detector layer.

FIG. 11 schematically illustrates an exemplary array of integratedanalytical devices, where each device comprises an absorptive filterlayer within the reflective cone, and there is no color separation inthe detector layer.

FIG. 12 schematically illustrates an exemplary array of integratedanalytical devices, where each device comprises an absorptive filterlayer within the reflective cone, and where the detector layer includesa two-color separation filter stack.

FIG. 13 schematically illustrates an exemplary ZMW module.

FIG. 14 schematically illustrates an exemplary array of integratedanalytical devices, highlighting parameters relating to the waveguidemodule layer.

FIG. 15 schematically illustrates an exemplary array of integratedanalytical devices, highlighting parameters relating to the collectionmodule layer and reflective cones.

FIG. 16 schematically illustrates an exemplary array of integratedanalytical devices, highlighting parameters relating to the filtermodule layer.

FIG. 17 schematically illustrates an exemplary array of integratedanalytical devices, highlighting parameters relating to the deep trenchopening module.

FIGS. 18A and 18B illustrate an exemplary process flow for themanufacture of an array of integrated analytical devices comprising adielectric filter module.

FIGS. 19A and 19B illustrate an exemplary process flow for themanufacture of an array of integrated analytical devices comprising anabsorptive filter module.

FIG. 20 illustrates an exemplary process flow variant for themanufacture of an array of integrated analytical devices comprising a2-color separation filter in the detector layer.

DETAILED DESCRIPTION OF THE INVENTION

Integrated Analytical Devices

Multiplexed optical analytical systems are used in a wide variety ofdifferent applications. Such applications can include the analysis ofsingle molecules, and can involve observing, for example, singlebiomolecules in real time as they carry out reactions. For ease ofdiscussion, such multiplexed systems are discussed herein in terms of apreferred application: the analysis of nucleic acid sequenceinformation, and particularly, single molecule nucleic acid sequenceanalysis. Although described in terms of a particular application, itshould be appreciated that the applications for the devices and systemsdescribed herein are of broader application.

In the context of single molecule nucleic acid sequencing analyses, asingle immobilized nucleic acid synthesis complex, comprising apolymerase enzyme, a template nucleic acid, whose sequence one isattempting to elucidate, and a primer sequence that is complementary toa portion of the template sequence, is observed to identify individualnucleotides as they are incorporated into the extended primer sequence.Incorporation is typically monitored by observing an opticallydetectable label on the nucleotide, prior to, during or following itsincorporation. In some cases, such single molecule analyses employ a“one base at a time approach”, whereby a single type of labelednucleotide is introduced to and contacted with the complex at a time.Upon incorporation, unincorporated nucleotides are washed away from thecomplex, and the labeled incorporated nucleotides are detected as a partof the immobilized complex.

In some instances, only a single type of nucleotide is added to detectincorporation. These methods then require a cycling through of thevarious different types of nucleotides (e.g., A, T, G and C) to be ableto determine the sequence of the template. Because only a single typenucleotide is contacted with the complex at any given time, anyincorporation event is by definition, an incorporation of the contactednucleotide. These methods, while somewhat effective, generally sufferfrom difficulties when the template sequence includes multiple repeatednucleotides, as multiple bases may be incorporated that areindistinguishable from a single incorporation event. In some cases,proposed solutions to this issue include adjusting the concentrations ofnucleotides present to ensure that single incorporation events arekinetically favored.

In other cases, multiple types of nucleotides are added simultaneously,but the nucleotides are distinguishable by the presence on each type ofnucleotide of a different optical label. Accordingly, such methods canuse a single step to identify a given base in the sequence. Inparticular, all four nucleotides, each bearing a distinguishable label,is added to the immobilized complex. The complex is then interrogated toidentify which type of base was incorporated, and as such, the next basein the template sequence.

In some cases, these methods only monitor the addition of one base at atime, and as such, they (and in some cases, the single nucleotidecontact methods) require additional controls to avoid multiple basesbeing added in any given step, and thus being missed by the detectionsystem. Typically, such methods employ terminator groups on thenucleotide that prevent further extension of the primer once onenucleotide has been incorporated. These terminator groups are typicallyremovable, allowing the controlled re-extension after a detectedincorporation event. Likewise, in order to avoid confounding labels frompreviously incorporated nucleotides, the labeling groups on thesenucleotides are typically configured to be removable or otherwiseinactivatable.

In another process, single molecule primer extension reactions aremonitored in real-time, to identify the continued incorporation ofnucleotides in the extension product to elucidate the underlyingtemplate sequence. In such single molecule real time (or SMRT™)sequencing, the process of incorporation of nucleotides in apolymerase-mediated template dependent primer extension reaction ismonitored as it occurs. In preferred aspects, the template/polymeraseprimer complex is provided, typically immobilized, within an opticallyconfined region, such as a zero mode waveguide (ZMW), or proximal to thesurface of a transparent substrate, optical waveguide, or the like (seee.g., U.S. Pat. Nos. 6,917,726, and 7,170,050 and U.S. PatentApplication Publication No. 2007/0134128, the full disclosures of whichare hereby incorporated by reference herein in their entirety for allpurposes). The optically confined region is illuminated with anappropriate excitation radiation for the fluorescently labelednucleotides that are to be used. Because the complex is within anoptically confined region, or very small illumination volume, only thereaction volume immediately surrounding the complex is subjected to theexcitation radiation. Accordingly, those fluorescently labelednucleotides that are interacting with the complex, e.g., during anincorporation event, are present within the illumination volume for asufficient time to identify them as having been incorporated.

A schematic illustration of this sequencing process is shown in FIG. 1.As shown in FIG. 1A, an immobilized complex 102 of a polymerase enzyme,a template nucleic acid and a primer sequence are provided within anobservation volume (as shown by dashed line 104) of an opticalconfinement, of e.g., a zero mode waveguide 106. As an appropriatenucleotide analog, e.g., nucleotide 108, is incorporated into thenascent nucleic acid strand, it is illuminated for an extended period oftime corresponding to the retention time of the labeled nucleotideanalog within the observation volume during incorporation which producesa signal associated with that retention, e.g., signal pulse 112 as shownby the A trace in FIG. 1B. Once incorporated, the label that attached tothe polyphosphate component of the labeled nucleotide analog, isreleased. When the next appropriate nucleotide analog, e.g., nucleotide110, is contacted with the complex, it too is incorporated, giving riseto a corresponding signal 114 in the T trace of FIG. 1B. By monitoringthe incorporation of bases into the nascent strand, as dictated by theunderlying complementarity of the template sequence, long stretches ofsequence information of the template can be obtained.

The above sequencing reaction may be incorporated into a device,typically an integrated analytical device, that provides for thesimultaneous observation of multiple sequencing reactions, ideally inreal time. While the components of each device and the configuration ofthe devices in the system may vary, each integrated analytical devicetypically comprises, at least in part, the general structure shown as ablock diagram in FIG. 2. As shown, an integrated analytical device 200typically includes a reaction cell 202, in which the reactants aredisposed and from which the optical signals emanate. The analysis systemfurther includes a detector element 220, which is disposed in opticalcommunication with the reaction cell 202. Optical communication betweenthe reaction cell 202 and the detector element 220 may be provided by anoptical train 204 comprised of one or more optical elements generallydesignated 206, 208, 210 and 212 for efficiently directing the signalfrom the reaction cell 202 to the detector 220. These optical elementsmay generally comprise any number of elements, such as lenses, filters,gratings, mirrors, prisms, refractive material, or the like, or variouscombinations of these, depending upon the specifics of the application.By integrating these elements into a single device architecture, theefficiency of the optical coupling between the reaction cell and thedetector is improved. Examples of integrated analytical systems,including various approaches for illuminating the reaction cell anddetecting optical signals emitted from the reaction cell, are describedin U.S. Patent Application Publication Nos. 2012/0014837, 2012/0019828,and 2012/0021525, which are each incorporated by reference herein intheir entireties for all purposes.

Conventional analytical systems typically measure multiple spectrallydistinct signals or signal events and must therefore utilize complexoptical systems to separate and distinctly detect those different signalevents. The optical path of an integrated device may be simplified,however, by a reduction in the amount or number of spectrallydistinguishable signals that are detected. Such a reduction is ideallyeffected, however, without reducing the number of distinct reactionevents that can be detected. For example, in an analytical system thatdistinguishes four different reactions based upon four differentdetectable signal events, where a typical system would assign adifferent signal spectrum to each different reaction, and thereby detectand distinguish each signal event, in an alternative approach, fourdifferent signal events would be represented at fewer than fourdifferent signal spectra, and would, instead, rely, at least in part, onother non-spectral distinctions between the signal events.

For example, a sequencing operation that would conventionally employfour spectrally distinguishable signals, e.g., a “four-color” sequencingsystem, in order to identify and characterize the incorporation of eachof the four different nucleotides, would, in the context of analternative configuration, employ a one-color or two-color analysis,e.g., relying upon a signals having only one or two distinct ordistinguished spectral signals. However, in such an alternativeconfiguration, this reduction in reliance on signal spectral complexitydoes not come at the expense of the ability to distinguish signals frommultiple, i.e., a larger number of different signal producing reactionevents. In particular, instead of relying solely on signal spectrum todistinguish reaction events, such an alternative configuration may relyupon one or more signal characteristics other than emission spectrum,including, for example, signal intensity, excitation spectrum, or bothto distinguish signal events from each other.

In one particular alternative configuration, the optical paths in anintegrated analytical device may thus be simplified by utilizing signalintensity as a distinguishing feature between two or more signal events.In its simplest iteration, and with reference to an exemplary sequencingprocess, two different types of nucleotides would bear fluorescentlabels that each emit fluorescence under the same excitationillumination, i.e., having the same or substantially overlappingspectral band, and thus would provide benefits of being excited using asingle excitation source and beam. The resulting signals from eachfluorescent label would have distinct signal intensities or amplitudesunder that same illumination, and would be distinguishable by theirrespective signal amplitudes. These two signals could have partially orentirely overlapping emission spectra, but separation of the signalsbased upon any difference in emission spectrum would be unnecessary.

Accordingly, for analytical systems using two or more signal events thatdiffer in signal amplitude, the integrated analytical devices of suchsystems can readily benefit through the removal of some or all of thosecomponents that would normally be used to separate spectrally distinctsignals, such as multiple excitation sources and their associatedoptical trains, as well as the color separation optics, e.g., filtersand dichroics, for the signal events, which in many cases, requires atleast partially separate optical trains and detectors for eachspectrally distinct signal. As a result, the optical paths for theseintegrated analytical devices are greatly simplified, allowing placementof detector elements in closer proximity to reaction regions, andimproving overall performance of the detection process for thesedevices.

Provision of signal producing reactants that will produce differentsignal amplitudes under a particular excitation illumination profile maybe accomplished in a number of ways. For example, different fluorescentlabels may be used that present excitation spectral profiles thatoverlap but include different maxima. As such, excitation at a narrowwavelength will typically give rise to differing signal intensities foreach fluorescent group. This is illustrated in FIG. 3A, which shows theexcitation spectra of two different fluorescent label groups (solid anddashed lines 302 and 304, respectively). When subjected to excitationillumination at the wavelength range shown by vertical lines 306, eachfluorescent label will emit a signal at the corresponding amplitude. Theresulting signal intensities at a given excitation wavelength are thenshown in the bar chart of FIG. 3B, shown as the solid lined and dashedlined bars, respectively. The difference in intensity of these twosignal producing labels at the given excitation wavelength can then bereadily used to distinguish the two signal events. As will beappreciated, such spectrally indistinct signals would not be easilydistinguishable when occurring simultaneously, as they would result inan additive overlapping signal, unless, as discussed below, suchspectrally indistinct signals result from spectrally distinct excitationwavelengths. As will be appreciated, this same approach may be used withmore than two label groups where the resulting emission at a givenexcitation spectrum have distinguishable intensities or amplitudes.

Similarly, two different fluorescent labeling groups may have the sameor substantially similar excitation spectra, but provide different anddistinguishable signal emission intensities due to the quantum yield ofthose labeling groups.

Further, although described in terms of two distinct fluorescent dyes,it will be appreciated that each different labeling group may eachinclude multiple labeling molecules. For example, each reactant mayinclude an energy transfer dye pair that yields emissions of differingintensities upon excitation with a single illumination source. Forexample, a labeling group may include a donor fluorophore that isexcited at a given excitation wavelength, and an acceptor fluorophorethat is excited at the emission wavelength of the donor, resulting inenergy transfer to the acceptor. By using different acceptors, whoseexcitation spectra overlap the emission spectrum of the donor todiffering degrees, such an approach can produce overall labeling groupsthat emit at different signal amplitudes for a given excitationwavelength and level. Likewise, adjusting the energy transfer efficiencybetween the donor and acceptor will likewise result in differing signalintensities at a given excitation illumination.

Alternatively, different signal amplitudes may be provided by differentmultiples of signal producing label groups on a given reactant, e.g.,putting a single label molecule on one reactant while putting 2, 3, 4 ormore individual label molecules on a different reactant. The resultingemitted signal will be reflective of the number of labels present on areactant and thus will be indicative of the identity of that reactant.

Exemplary compositions and methods relating to fluorescent reagents,such as nucleotide analogs, useful for the above purposes are describedin, for example, U.S. Patent Application Publication Nos. 2012/0058473;2012/0077189; 2012/0052506; 2012/0058469; 2012/0058482; 2010/0255488;2009/0208957, which is each incorporated by reference herein in itsentirety for all purposes.

As described above, integrated analytical devices making use of suchapproaches see a reduction in complexity by elimination of spectraldiscrimination requirements, e.g., using signal amplitude or othernon-spectral characteristics as a basis for signal discrimination.Integrated analytical devices that combine such non-spectraldiscrimination approaches with the more common spectral discriminationapproaches may also provide advantages over more complex spectraldiscrimination systems. By shifting from a “four-color” discriminationsystem to a system that distinguishes signals based upon signalintensity and color, one can still reduce the complexity of the overalloptical system relative to a conventional four-color separation scheme.For example, in an analytical operation that detects four discretereaction events, e.g., in a nucleic acid sequencing analysis, two signalevents may be provided within a given emission/detection spectrum, i.e.,emitting signals within the same spectral window, and the other twoevents within a distinct emission/detection spectrum. Within eachspectral window, the pair of signal events produce distinguishablesignal intensities relative to each other.

For ease of discussion, this concept is described in terms of two groupsof fluorescent signal events, where members of each group differ byfluorescent intensity, and the groups differ by virtue of their emissionspectrum. As will be appreciated, the use of simplified optics systems,e.g., using two detection channels for two distinct emission spectra,does not require that the emission profiles of the two groups of signalsdo not overlap or that the emission spectra of members of each groupperfectly overlap. Instead, in many preferred aspects, more complexsignal profiles may be used where each different signal event possessesa unique emission spectrum, but in a way that each signal will present asignal profile within the two detection channels that is unique, basedupon the signal intensity in each channel.

FIG. 4 schematically illustrates the signal profiles for each of fourfluorescent labeling groups, overlain with each of two different filterprofiles. As shown, four label groups yield emission spectra 402, 404,406 and 408, respectively. While the signals from these four groupspartially overlap each other, they each have different maxima. Whensubjected to a two channel filter scheme, as shown by pass filter lines410 and 412, the signal from each label will produce a unique signalprofile between the two detection channels. In particular, signals arerouted through an optical train that includes two paths that arefiltered according to the spectral profile shown. For each signal,different levels of emitted light will pass through each path and bedetected upon an associated detector. The amount of signal that passesthrough each filter path is dictated by the spectral characteristics ofthe signal.

FIG. 5 illustrates modeled signal data plotted as a function of detectedchannel 1 and channel 2 intensity. As can be seen, signals 402, 404, 406and 408 associated with each different group presents a unique signalprofile that is a combination of channels 1 and 2 intensity. Inparticularly preferred aspects, each of the label groups orsignal-producing reactants that is sought to be distinguished using theschemes described herein, is selected to be sufficiently different fromeach other label in at least one of the two detection channels so as tobe distinguishable from each other signal based upon a combination ofsignals from each of the two detection channels.

In the case of the above described mixed-mode schemes, detection systemsmay be provided that include at least two distinct detection channels,where each detection channel passes light within a spectrum that isdifferent from each other channel. Such systems also include a reactionmixture within optical communication of the detection channels, wherethe reaction mixture produces at least three different optical signalsthat each produces a unique signal pattern within the two detectionchannels, as compared to the other optical signals.

In each case, each signal-producing reactant is selected to provide asignal that is entirely distinct from each other signal in at least oneof signal intensity and signal channel. As noted above, signal intensityin a given channel is dictated, in part, by the nature of the opticalsignal, e.g., its emission spectrum, as well as the filters throughwhich that signal is passed, e.g., the portion of that spectrum that isallowed to reach the detector in a given channel. However, signalintensity can also be modulated by random variables, such as orientationof a label group when it is emitting signal, or other variables of theparticular reaction. Accordingly, for a signal's intensity to be assuredof being entirely different from the intensity of another signal withina given channel, in preferred aspects, this variation is accounted for.

With a reduced number of spectrally distinct signal events, thecomplexity of the optical paths for the integrated devices is alsoreduced. FIG. 6 illustrates a not-to-scale example device architecturefor performing optical analyses, e.g., nucleic acid sequencingprocesses, that rely in part on non-spectral discrimination of differingsignals, and optionally, in part on spectral distinction. As shown, anintegrated analytical device 600 includes a reaction region 602 that isdefined upon the surface layer of the device. As shown, the reactionregion comprises a nanowell disposed in the surface layer. Suchnanowells may constitute depressions in a substrate surface or aperturesdisposed through additional substrate layers to an underlyingtransparent substrate, e.g., as used in zero mode waveguide arrays (See,e.g., U.S. Pat. Nos. 7,181,122 and 7,907,800).

Excitation illumination is delivered to the reaction region from anexcitation light source (not shown) that may be separate from or alsointegrated into the substrate. As shown, an optical waveguide (orwaveguide layer) 606 may be used to convey excitation light (shown byarrows) to the reaction region/nanowell 602, where the evanescent fieldemanating from the waveguide 606 illuminates reactants within thereaction region 602. Use of optical waveguides to illuminate reactionregions is described in e.g., U.S. Pat. No. 7,820,983 and U.S. PatentApplication Publication No. 2012/0085894, which are each incorporated byreference herein in their entireties for all purposes. The nanowell actsto enhance the emission of fluorescence downward into the device andlimit the amount of light scattered upwards.

The emitted light is directed into the device through an integratedoptical train 604 comprising one or more optical elements. The opticaltrain optionally includes light channeling components 608 to efficientlydirect emitted light from the reaction regions to a detector layer 612disposed beneath the reaction region. As described in more detail below,the collection path may include reflective cones and/or optical lensesto channel the emitted light and/or to split the light into multiplebeams. The optical lenses within the collection path may be refractivelenses but are preferably diffractive lenses. The lenses may, forexample, split the emitted light into two, three, four, or even morebeams directed onto the detector layer. The split beams may be organizedin a linear fashion, or they may be arranged in an array, for example ina 2×2 beam array or the like, depending on the configuration of thedetector elements.

The detector layer typically comprises one, or preferably multiple,detector elements 612 a-d, e.g., pixels in an array detector, that areoptically coupled to a given reaction region. Although illustrated as alinear arrangement of pixels 612 a-d, it will be appreciated that suchpixels may be arranged in a grid, n×n square, n×m rectangle, annulararray, or any other convenient orientation.

Emitted signals from the reaction region 602 that impinge on thesepixels are then detected and recorded. As noted above, an optionalsingle filter layer 610 is disposed between the detector layer and thereaction region, to permit different spectrally distinct signals totravel to different associated pixels 612 a and 612 b in the detectorlayer 612. For example, the portion 610 a of filter layer 610 allowssignals having a first emission spectrum to reach its associated pixels612 a and 612 b, while filter portion 610 b of filter layer 610 allowsonly signals having a distinct second spectrum to reach its associatedpixels 612 c and 612 d.

In the context of a sequencing system exploiting such a configuration,incorporation of two of the four nucleotides would produce signals thatwould be passed through filter portion 610 a to pixels 612 a and 612 b,and blocked by filter portion 610 b. As between these two signals, onesignal would have a signal intensity higher than the other such that thepixels 612 a and 612 b in detector layer 612 would be able to producesignal responses indicative of such differing signal intensities.Likewise, incorporation of the other two nucleotides would producesignals that would be passed through filter portion 610 b to itsassociated pixels 612 c and 612 d, while filter portion 610 a wouldblock those signals from reaching pixels 610 a and 610 b. Again, thesignals associated with these two latter signal events would differbased upon their signal intensities or amplitudes. In someconfigurations, for example if the amplitudes of the different dyes areproperly calibrated, it may alternatively be possible to differentiatefour different dyes using only two pixels in the detector layer.

The detector layer is then operably coupled to an appropriate circuitry,typically integrated into the substrate, for providing a signal responseto a processor that is optionally included integrated within the samedevice structure or is separate from but electronically coupled to thedetector layer and associated circuitry. Examples of types of circuitryare described in U.S. Patent Application Publication No. 2012/0019828.

As will be appreciated from the foregoing disclosure and FIG. 6, theintegrated analytical devices described herein do not require the morecomplicated optical paths that are necessary in systems utilizingconventional four-color optics, obviating the need for excessive signalseparation optics, dichroics, prisms, or filter layers. In particular,although shown with a single filter layer, as noted, in optionalaspects, the filter layer could be eliminated or could be replaced witha filter layer that blocks stray light from the excitation source ratherthan distinguishing different emission signals from the reaction region.Even including the filter layer 610, results in simplified and/or moreefficient optics as compared to conventional four-color systems, whichwould require either multilayer filters, or narrow band pass filters,which typically require hybrid layers or composite approaches over eachsubset of pixels, thus blocking signal from reaching three of the fourpixel subsets at any given emission wavelength, resulting in detectionof far fewer photons from each signal event. The optics configurationshown in FIG. 6, on the other hand, only blocks a smaller portion of theoverall signal light from reaching the detector. Alternatively, suchconventional systems would require separation and differential directionof all four different signal types, resulting in inclusion of additionaloptical elements, e.g., prisms or gratings, to achieve spectralseparation.

FIG. 7 shows a schematic exemplar signal output for a real timesequencing operation using a two color/two amplitude signal set from anintegrated system of the invention where one trace (dashed) denotessignals associated with incorporation of A (high intensity signal) and T(lower intensity signal) bases, while the other signal trace (solidline), denotes the signals of a different emission spectrum, associatedwith G (high) and C (low) bases. The timing of incorporation and theidentity of the base incorporated, as derived from the color channel andintensity of the signal, are then used to interpret the base sequence.

The process flows disclosed as part of the instant invention provide forthe production of novel filter architectures. In particular, as notedabove, typical four-color detection schemes operate through thedetection of signal at a narrow spectral band corresponding to andcorrelating with an emission signal maximum emitted from a particularreaction event, e.g., incorporation of a single type of nucleotide in asequencing operation, with the remainder of the spectrum being blockedand disregarded. In the context of single molecule analyses and/or smallscale integrated devices, however, where signal detection efficiency isof far greater importance, discarding of any photons associated with aparticular reaction event should be avoided as much as possible.

Accordingly, in certain aspects, the present invention provides arraysof integrated analytical devices for use in optical detection systemsthat reduce the attenuation of optical signals emanating from thereaction region and ultimately, that reach the detector. This permitsdetection and signal discrimination that is based upon a greater amountof emitted and detected signal.

One approach to this aspect of such optical detection systems is ideallyillustrated in the context of multicolor fluorescence detection systems,e.g., the four-color fluorescence systems described above. As noted,typically, such systems include reactions that produce different opticalsignals based upon the occurrence of different reaction events, such asincorporation of different fluorescently labeled nucleotides in manynucleic acid “sequencing by synthesis” applications.

Signals that are indicative of the addition of a given base to thepolymerase/template/primer replication complex are typically passedthrough a series of optical filters that narrowly separate out eachsignal component based upon its spectral maximum, and direct thatseparated component to a separate detector or sensing region of adetector, e.g., a pixel or subset of pixels in an array detector. Thetype of base added in a given step is then identified from the narrowsignal component that is detected at that particular juncture in theassay. While this method is highly effective for many applications,where signal is very limited, e.g., where attempting to detect signalfrom a very small reaction volume or a single molecule of a fluorescentlabel, narrowly attenuating that signal becomes more problematic.

The potential difficulties are schematically illustrated in FIG. 8 withreference to an exemplary four-color DNA sequencing system. As shown inFIG. 8A, the signal palette for the four bases in an exemplary DNAsequencing reaction are shown as four distinct, albeit partiallyoverlapping emission maxima 802, 804, 806 and 808. In conventionalfour-color detection systems, signals from the reaction zone are passedthrough a filter system, typically comprised of multiple filters, thatallow a narrow spectral band, e.g., spectral band 810, 820, 830 or 840,that corresponds to an emission maximum for each differently labelednucleotide (e.g., A, T, G, and C, respectively) to reach one of fourdifferent detectors or detection zones on the same detector. Forconvenience, different detectors or different detection zones on thesame detector are interchangeably referred to herein as different“detectors”. FIG. 8B shows a schematic illustration of a signal profilefor each base, based upon such conventional systems. As shown, e.g., foran A base incorporation, a signal for a given base is substantially onlydetected upon detector 1, while being blocked from or significantlyattenuated at detectors 2, 3 and 4.

This technique is effective where signals of a given spectral band arecompletely separated from other signals and the separated signals aredirected to a detector where all light associated with that signal canbe detected. However, for miniaturized systems, the ability tocompletely separate different signals and detect all light associatedwith the separated components is impaired by the structural size of thedevices. In particular, signal “separation” in certain implementationsof integrated analytical devices may fractionate a signal and subjecteach fraction to a different filter set, in order to distinguishdifferent signals.

In accordance with an improved alternative integrated devicearchitecture, however, the filtering approach is inverted such that eachfilter for each of the different detectors or sensing elements wouldrepresent a narrow-band blocking filter disposed between the assayregion and the detector, that only blocks the indicated portion of thespectrum, e.g., spectral band 1, 2, 3 and 4 in FIG. 8A, from reachingits respective detector component. Accordingly, each signal results indetection at three of the four detection zones.

FIG. 9 schematically shows the signal profile at each of the detectorsfor all four bases. As can be seen, each signal is detected from photonsreaching three of the four detectors and that are only attenuated at asingle different detector by virtue of the narrow band blocking filter.As is also apparent, each signal event results in a greater amount ofdetected signal than would be provided in a signal profile from anarrow-band pass filter architecture, e.g., as illustrated in FIG. 8B,above. The resulting unique signal profile over multiple detectors canthen be used to identify the nature of the fluorescent label, andconsequently, the added base. In effect, such a filter scheme results ina “negative” of the signal profile from the conventional narrowband-pass scheme. Although described in terms of four-color schemes, itwill be appreciated that this approach can also be applied to fewer orgreater than four-color schemes, e.g., three-color schemes, five-colorschemes, or the like.

Described differently, each detector or detector region, e.g., pixelsubset on a given detector, has a filter layer that permits greater than25% of light from the totality of the various different optical signalsimpinging upon the filter layer to pass through to the detector or pixelsubset. In some configurations, that filter layer will permit greaterthan 50% of light from the totality of the various different opticalsignals impinging upon the filter layer to pass through the detector orpixel subset, and in additional configurations, greater than 60% of thelight that impinges on the filter from the totality of optical signalswill pass through a given filter layer to reach its associated pixelsubset, and in some cases, greater than 70% of the light that impingeson the filter from the totality of optical signals will pass through agiven filter layer to reach its associated pixel subset.

In addition to benefits of increased signal at each detector, thisaspect of an improved alternative device architecture providesadditional benefits in the context of integrated optical devices, e.g.,devices in which at least the optical components, e.g., filters and thelike, and detector elements are integrated into a single substrate. Inparticular, by providing a single narrow-band blocking filter type,rather than a filter stack between the assay location and a givendetector, the overall architecture of the device can be greatlysimplified. In particular, by using only a single narrow band blockingfilter, it is possible to use single layers or single composition filterlayers rather than hybrid filter layers or filter compositions.

Further, because fewer layers are provided between the assay region andthe detector, the assay location can be provided in closer proximity tothe detector, reducing the potential for signal loss, cross-talk, andother signal transmission difficulties that may be inherent in morecomplex optical trains. In particular, where a more conventionalfour-color system might require a four-layer optical filter at eachdetector, and result in a substantially attenuated signal, the systemsdescribed herein would include a single filter layer at each detector,and result in the higher level signal profiles described above.

The devices and systems disclosed herein may generally be characterizedby virtue of the number of filter layers as it relates to the number ofspectrally distinct optical signals to be detected.

In yet another alternative approach, the integrated analytical systemsdisclosed herein simplify the optical path by relying on assay processesthat utilize other than spectral separation of different signals todistinguish different signal events. Examples of such temporal signaldistinction are described in U.S. Patent Application Publication Nos.2012/0019828 and 2009/0181396, incorporated by reference herein in theirentireties for all purposes, and relies upon the use of differentfluorescent labeling groups that possess distinct excitation spectra. Bymodulating the excitation light through each of the different excitationspectra, and correlating any resulting emitted fluorescence with theexcitation spectrum at a given time, one can identify what excitationlight caused a given emission, and consequently identify the fluorescentlabel and the reaction or reagent with which it is associated. As willbe appreciated, this type of excitation and detection scheme requires nosignal filtering optics, other than as necessary to screen outbackground or other incidental light, e.g., excitation illumination.

In the context of a number of aspects of the systems disclosed herein,for systems that have greater than 2, greater than 3 or greater than 4or more, spectrally distinct optical signals, the system will include afilter component that rejects or attenuates fewer than n−1 of thosedistinct optical signals, where n is the number of spectrally distinctsignals, e.g., signals associated with different fluorescent label setsor different labeled reactants or reaction products. For example, withreference to the scheme described for FIG. 6, above, a single signalattenuating filter, e.g., filter layer portion 610 a in FIG. 6, may beused between the reaction region and a given detector, with a differentsingle attenuating filter, e.g., filter layer portion 610 b in FIG. 6,being provided over each of the four different detectors. Likewise, fora two-color, two signal intensity signal profile for a given analysis,again a single signal attenuating filter element is provided over two ofthe detectors and a different single signal attenuating filter isprovided over the other two detectors.

Arrays of Integrated Analytical Devices

In order to obtain the volumes of sequence information that may bedesired for the widespread application of genetic sequencing, e.g., inresearch and diagnostics, higher throughput systems are desired. By wayof example, in order to enhance the sequencing throughput of the system,multiple complexes are typically monitored, where each complex issequencing a separate template sequence. In the case of genomicsequencing or sequencing of other large DNA components, these templateswill typically comprise overlapping fragments of the genomic DNA. Bysequencing each fragment, one can then assemble a contiguous sequencefrom the overlapping sequence data from the fragments.

As described above, and as shown in FIG. 1, the template/DNApolymerase-primer complex of such a sequencing system is provided,typically immobilized, within an optically confined region, such as azero mode waveguide (ZMW), or proximal to the surface of a transparentsubstrate, optical waveguide, or the like. Preferably, such reactioncells are arrayed in large numbers upon a substrate in order to achievethe scale necessary for genomic or other large-scale DNA sequencingapproaches. Such arrays preferably comprise a complete integratedanalytical device, such as, for example, the devices shown in the blockdiagrams of FIGS. 2 and 6. Examples of integrated systems comprisingarrays of optical analytical devices are provided in U.S. PatentApplication Publication Nos. 2012/0014837; 2012/0019828; and2012/0021525.

Arrays of integrated analytical devices, such as arrays of devicescomprising ZMWs, can be fabricated at ultra-high density, providinganywhere from 1000 ZMWs per cm², to 1,000,000 ZMWs per cm², or more.Thus, at any given time, it may be desirable to analyze the reactionsoccurring in from 100, 1000, 3000, 5000, 10,000, 20,000, 50,000, 100,000or 1 Million, 10 Million or more ZMWs or other reaction regions within asingle analytical system or even on a single substrate.

Using the foregoing systems, simultaneous targeted illumination ofthousands or tens of thousands of ZMWs in an array has been described.However, as the desire for multiplex increases, the density of ZMWs onan array, and the ability to provide targeted illumination of sucharrays, increases in difficulty, as issues of ZMW cross-talk (signalsfrom neighboring ZMWs contaminating each other as they exit the array),decreased signal:noise ratios arising from higher levels of denserillumination, and the like, increase. The arrays and methods of theinstant invention address some of these issues.

The position on the detector upon which a given signal is incident isindicative of (1) the originating ZMW in the array, and (2) the emissioncharacteristics of the signal component, which is used, for example, toidentify the type of fluorescently labeled nucleotide analogincorporated in an extension reaction. As noted above, the detector mayinclude in some cases multiple sensing elements, each for detectinglight having a different color spectrum. For example, in the case ofsequencing, the sensor for each reaction cell may have 4 elements, onefor each of the four bases. In some cases, the sensor elements providecolor discrimination, in other cases, color filters are used to directthe appropriate color of light to the appropriate sensor element. Insome cases, the sensor elements detect intensity of signal only, withoutdiscriminating color. In some cases, the sensor elements identify theincorporated nucleotide using a combination of emission characteristics.

Exemplary arrays of integrated analytical devices are illustratedschematically in FIGS. 10-12, wherein the repeating device unit of eacharray is represented as the area within the brackets in each drawing. Aswould be understood by one of ordinary skill in the art, the arrays ofthe instant invention can include any desired number and density of ZMWsby repetition of the individual analytical device unit in twodimensions, i.e., along the horizontal axis of the drawing and along theaxis extending perpendicular to the plane of the drawing. Suchrepetition of the individual analytical device unit allows thegeneration of two-dimensional arrays with extremely large numbers andultra-high densities, as described above. Fabrication of such arrays isdescribed in the following section using the methods of the instantdisclosure. The arrangement and placement of individual analyticaldevices within the two-dimensional array is achieved through thefabrication methods and can be modified as desired within the scope ofthe instant invention. In some cases, the analytical devices arearranged relative to one another in regular rows and columns, but otherarrangements may also be generated, if so desired, during thefabrication process.

The arrays of integrated analytical devices illustrated in FIGS. 10-12share several common features. For example, each integrated analyticaldevice within the array includes a ZMW module layer (1001, 1101, and1201) comprising a nanometer-scale aperture penetrating into the uppercladding of the waveguide module layer (1002, 1102, and 1202). Thewaveguide module layer comprises a core of high refractive index (“highn”) and a cladding of low refractive index (“low n”) that encapsulatesthe core. Examples of waveguides useful in the waveguide module layersof the instant application are disclosed in U.S. Pat. No. 7,820,983 andU.S. Patent Application Publication No. 2012/0085894. As would beunderstood by one of ordinary skill in the art, the waveguide modulelayer may propagate excitation illumination to the ZMWs of the array ina specific pattern, for example through a series of channels within thewaveguide module layer, or may propagate the illuminationnon-specifically in two dimensions, for example through a slab of corematerial that is defined within the cladding of the waveguide modulelayer.

The individual analytical devices within an array typically furtherinclude a collection module layer (1003, 1103, and 1203) and a filtermodule layer (1004, 1104, and 1204), which are disposed between the ZMWand the detector layer (1005, 1105, and 1205), just below the waveguidemodule layer. The collection module layer and the filter module layerare preferably fashioned in a cone shape that is defined by a reflectivelayer (1007, 1107, and 1207) covering the sides of the collection modulelayer and filter module layer but providing an opening for emitted lightto pass from the ZMW to the detector.

The arrays of integrated analytical devices may optionally furtherinclude features on the periphery of the array, such as a deep trench(1006, 1106, 1206), an optical coupler (1008, 1108, and 1208), and analignment feature (1009, 1109, 1209). These features are typically notrepeated in each of the individual analytical device units but may beseparately repeated as part of an array, for example in severallocations on a wafer, if so desired. These features may, for example,facilitate the assembly of a completed array into a larger analyticaldevice, or provide for a connection pathway between the top surface andthe bottom surface of the array (e.g., the alignment feature and thedeep trench) or may provide for a connection between a light source andthe waveguide module layer (e.g., the optical coupler). As is understoodin the art, proper alignment of the optical features within eachintegrated analytical device is critical in the effective function of anintegrated analytical device. Alignment features, such as for examplethose just described, may be used to effect or facilitate suchalignment. Other components may include packaging components, e.g.,components that provide fluidic interfaces with the surface of thearray, such as flow cells, wells or recesses, channel networks, or thelike, as macrostructures as compared to the surface defined structuresabove, as well as alignment structures and casings that providestructural protection for the underlying arrays and interactivefunctionality between the arrays and instrument systems that workwith/analyze the arrays. Other such optional features may be included inthe arrays without deviating from the overall scope of the invention.

The arrays of integrated analytical devices illustrated in FIGS. 10-12differ in the nature of their filter module layers and in the optionalinclusion of a color-separation thin-film stack layer as part of thedetector layer. Specifically, the filter module layer of the arrayillustrated in FIG. 10 comprises a dielectric filter layer within thereflective cone, whereas the filter module layer of the arrayillustrated in FIG. 11 comprises an absorptive filter layer within thereflective cone. Neither of these examples includes any color-separationlayers. The devices of these arrays therefore correspond most similarlyto the device of FIG. 6, with a single filter layer 610 (i.e., 610 a and610 b are the same material) and with a single detector element (i.e.,612 a-d do not distinguish color). As described above, such devices relyon features of the emission signal other than spectral differences toidentify incorporation events The filter module layer of the arrayillustrated in FIG. 12 comprises an absorptive filter layer within thereflective cone, and further includes a 2-color separation thin-filmstack layer as part of the detector layer. The devices of this arraytherefore correspond most similarly to the device of FIG. 6, with asingle filter layer 610 (i.e., 610 a and 610 b are the same material)and with a two-color detector element (i.e., the detector elements 612 aand b are combined into a single element, and the detector elements 612c and d are combined into a single element, and the two combinedelements have different color sensitivity). As described above, suchdevices rely both on the differences in the emission spectrum and onfeatures of the emission signal other than spectral differences toidentify incorporation events. As would be understood by one of ordinaryskill in the art, the arrays illustrated in FIGS. 10-12 could be furthermodified to include alternative filter layers and detector elements,such as those shown in FIG. 6.

As noted above, the ZMWs of the devices of the instant arrays arenanometer-scale apertures that penetrate into the upper cladding of thewaveguide module layer. For example, FIG. 13 illustrates an exemplaryZMW, wherein the ZMW material (M1) is deposited on the surface of thelow refractive index (“low n”) waveguide cladding layer (M2). M1 istypically a highly reflective metal and in preferred embodiments isaluminum. M2 is typically SiO₂ or another equivalent low n dielectricmaterial. Distance d₂ is preferably from 50 to 150 nm and is mostpreferably approximately 100 nm. Distance d can range from 0 to 200 nm.In preferred embodiments, d is 100 nm±10%. In most preferred embodimentsof the invention, d is 100 nm. The preferred angles of α1 and α2 are101° and 95°, but other reasonable angles are within the scope of theinvention. The ZMW pitch (the spacing between individual ZMWs) ispreferably 6.7 um, but other reasonable values are within the scope ofthe invention. Specifically, the pitch may range from 2.5 to 8 um,depending on the camera and pixel selection. In addition, the overlay tocone (x,y,θ) is most preferably 150 nm, but a range of 20 to 200 nm iswithin the scope of the invention. Values for the distances A, B, and Care in some embodiments 200 nm, 160 nm, and 140 nm±15%. In preferredembodiments, the variability in these distances is as low as ±5%. Theinset drawing on the right side of FIG. 13 shows a schematic view of atypical ZMW, as viewed from above.

More specific details relating to the waveguide module layer of theinstant arrays of integrated analytical devices are illustratedschematically in FIG. 14. Specifically, distances X₁, X₂, and X₃ arepreferably 500 nm, 1000 nm, and 1200 nm, respectively, but values of 400to 1500 nm for each of these parameters are considered within the scopeof the invention. Distance d is preferably 50 nm but in some embodimentscan range from 50 to 100 nm. The value for w (width into plane) ispreferably 300 nm but can be as high as 1 um. The parameter, R, is thetotal range of waveguide thickness variation, which includes thethickness variation, side wall roughness of the waveguide (short scale<500 nm length), and the long range roughness of the waveguide (wavinessat >10 um range). The overlay to ZMW (y, θ) is preferably 100 nm.

The material comprising the waveguide core, M3, is preferably siliconnitride (Si₃N₄, n(1.9)), but other materials, such as Al₂O₃ or othermaterials with high n (refractive index) and low autofluorescence areconsidered within the scope of the invention. As mentioned above, thewaveguide core can be configured either in channels or as a planarwaveguide, where the excitation illumination is propagated through theplane of the waveguide in two dimensions.

Parameters relating to the fabrication of the collection layer moduleand reflective cones are illustrated schematically in FIG. 15.Specifically, the distances c₁, c₂, and c₃ are preferably 6 um, 3 um,and 5.6 um, respectively, but other reasonable values for and variationin these distances are within the scope of the invention. The spacing,P, between ZMWs is preferably 6.7 um, but other reasonable values shouldbe considered within the scope of the invention. Thickness t is 200 nm,but variation within this value is also understood to be within thescope of the invention. The material used in the reflective layer of thecone, M4, is preferably Al, but other reflective materials may besuitably substituted for this purpose. Likewise, the filler materialwithin the cone, M5, and outside the cone, M5′, is preferably an oxideor other suitable material, provided that the material is a lowrefractive index (low n) material and has low autofluorescence. M5 andM5′ can be the same or different materials.

FIG. 16 provides a schematic illustration of the two preferred types offilter module layers: (A) a dielectric filter stack module and (B) anabsorptive filter module. Panel (C) shows the composition of the filterstacks within the cones of the filter module layer of panel (A).Specifically, the filter stacks in the dielectric filter stack moduleare comprised of alternative layers of either GaP and TiO₂ or TiO₂ andSiO₂. For the GaP/TiO₂ design, the number of layers (n) is preferably31, and the total thickness is preferably 1.2 um. The layer thicknessesare preferably 38 nm (th1) and 59 nm (th2), and the top (n+1) layerthickness is preferably 1.8 um (th3). For the TiO₂/SiO₂ design, thenumber of layers (n) is preferably 71, and the total thickness ispreferably 6 um. The layer thicknesses are preferably 48 nm (th1) and 31nm (th2). The long-range roughness, R₂, is preferably <5 nm. Theabsorptive filter of the filter layer module shown in the array of panel(B) is preferably composed of KMPR MicroChem PR with total layers, n=½um and Th1: 2 um; Th2: 3 um, but other materials with similar propertiescould be suitably substituted therefor. In all cases, the purpose of thefilter module layer is to cut out stray light from the excitationillumination, typically any light at or below 532 nm in wavelength, andallow emission light above this wavelength to pass with minimal loss.Absent any color separation in the detector layer, the incorporationsignals from the ZMW can thus be read by amplitude modulation or othernon-spectral discrimination, as described in detail above.

The deep trench opening module (1006, 1106, and 1206 of FIGS. 10-12,respectively) is illustrated schematically in FIG. 17. The trench fillmaterial is preferably Al or Cu, but other suitable materials may besubstituted. The deep trench can either be filled and then wire bondedor directly put through wire bonding using the above materials. Thelower (O₁) and upper (O₂) dimensions are preferably 100 um and 150 um,but reasonable variation in these values is possible within the scope ofthe invention. The deep trench opening usefully provides access to thebond pads for the CMOS sensor.

Controlled ZMW Nanowell Dimensions

In another aspect, the instant disclosure provides arrays of integratedanalytical devices wherein the dimensions of the ZMW nanowells arecontrolled by etching the upper cladding of the ZMW module layer untilthe nanowells fully penetrate into the upper cladding of the waveguidemodule layer, and then partially backfilling the etched nanowells. Alsoprovided are methods for such control of the dimensions of the ZMWnanowells.

As previously described, the hole pattern for the ZMW nanowells istypically defined using an appropriate lithography tool, such as a 193nm or 248 nm lithography node, and a photoresist material. Ideally, thenanowell is aligned as closely as possible to the underlying cone, orother collection feature, to ensure efficient transfer of emitted lightsignals from the ZMW to the associated detector feature and to minimizecross-talk. In some method embodiments disclosed herein, the nanowellsare created using a timed reactive ion etch (RIE) on the patternedwafer. The RIE process is allowed to proceed for a predefined amount oftime that is targeted to produce nanowells with a specified depth anddistance from the underlying waveguides. Process variability in the RIE,as well as in the upstream processes, such as the cladding deposition,may, in some situations, result in a variation in the nanowell depth anddistance from the waveguide, however, and alternative methods forcontrolling the dimension of the nanowell may be desirable in somecircumstances.

Accordingly, in order to minimize variability in the nanowelldimensions, the nanowells may, in some embodiments, be constructed byallowing the RIE to proceed until the etching process fully penetratesthe cladding material and exposes the waveguide. At this point, due tothe difference in chemical composition of the waveguide compared to thecladding, the exposure of the waveguide material to the reaction chambermay generate an endpoint signal that is detectable by the processingtool and that can be used to terminate the RIE. Suitable selection ofcladding and waveguide materials prevents significant etching or gougingof the waveguide and provides a strong endpoint signal. For example, inpreferred embodiments, SiO₂ may be utilized for the cladding, and Si₃N₄may be utilized for the waveguide. In alternative embodiments, an etchhardmask may be placed between the cladding and waveguide layers duringthe deposition of the cladding and waveguide stack in order to providean endpoint signal or a resistance to the RIE, assuming that thehardmask used is optically compatible with the device. Suitablematerials for the etch hardmask are well-known in the art.

Upon completion of the nanowell RIE process and the subsequent steps toremove etch residue and to clean the wafer, the nanowells may bepartially backfilled with a well-controlled deposition process until thedesired nanowell depth and distance to the waveguide is achieved. Lowvariability backfill processes are well known in the art, and theirutilization may significantly reduce variances in the nanowell depth anddistance from the waveguide. For example, atomic layer deposition (ALD)and low pressure chemical vapor deposition (LPCVD) are processes thatcan be very tightly controlled. The backfill material is preferablychosen to match the desired optical characteristics of the device. Forexample, the backfill material may in some embodiments be identical tothe cladding material, although this is not a requirement. In addition,the pattern mask dimensions and as-etched nanowell dimensions arepreferably specified to match the method of backfilling in order toachieve the desired final nanowell dimensions. For example, the requiredmask and as-etched nanowell dimensions may differ for a “conformal”deposition process, as compared to a “bottom-up” deposition process, aswould be understood by those of ordinary skill in the art.

Lenses for Collimation and Redirection

In another aspect, the instant disclosure provides additional oralternative features within the collection module layer that improve thetransmission of emission light from the ZMW nanowell to the detector. Asdescribed above, the collection module layer and the filter module layerare preferably fashioned in a cone shape that is defined by a reflectivelayer (see 1007, 1107, and 1207 in FIGS. 10, 11, and 12, respectively).The reflective layer covers the sides of the collection module layer andfilter module layer of each integrated analytical device but provides anopening to allow emitted light to pass through. Such reflective conesimprove the efficiency of capture of emitted light and minimizecross-talk between adjacent devices, as described, for example, in U.S.Patent Application Publication No. 2010/0099100, which is incorporatedby reference herein in its entirety for all purposes. Parametersrelating to the collection module layer and the reflective cones areillustrated in FIG. 15.

In some embodiments, it may be desirable to integrate one or moreoptical lenses into the collection module layer, either in combinationwith, or in place of, the reflective cones. Such optical lenses mayserve to collimate and/or redirect light emitted from the ZMW. Inparticular, optical lenses are well suited for collimating emitted lightwith near on-axis rays, as well as splitting the emitted light, forexample prior to color separation within the filter module layer. Inaddition, lenses are readily fabricated using standard techniques.

The integrated optical lenses of the instant devices may be eitherrefractive lenses or diffractive lenses, depending on the optical andphysical properties desired, as would be understood by those of ordinaryskill in the art. Diffractive lenses may, in some circumstances, provideimproved image quality, be more easily miniaturized, and/or be lessexpensive to fabricate than a comparable refractive lens. In some cases,the lenses may include separate refractive and diffractive components ormay be hybrid lenses that combine both features in a single lenselement.

In preferred embodiments, the integrated lenses of the instantanalytical devices are integrated Fresnel lenses, which may also beknown as zone plates or Fresnel zone plates when they function bydiffraction rather than refraction or reflection. A Fresnel lensconsists of a series of concentric rings with a specific tapered shape,or with alternating transparent and opaque zones (also called theFresnel zones), with respect to the incident irradiation. Thesestructures result in the focusing of light passing through the device byselective absorption or selective phase shifting and thus allow thedevice to function as a lens. The specific device design depends on theradiation to be focused, the refractive index of the material used toconstruct the lens, and the desired focal length, as is well known inthe art. In some embodiments, the Fresnel lenses of the instant devicesare refractive Fresnel lenses, in some embodiments, the Fresnel lensesare diffractive Fresnel lenses (or Fresnel zone plates), and in someembodiments, the Fresnel lenses combine refractive and diffractivefeatures. In preferred embodiments, the Fresnel lenses are diffractiveFresnel lenses.

A variety of materials and methods may be used to fabricate Fresnellenses, as is known in the art. For example, Fresnel lenses may beformed by the etching of zones in the planar surface of a materialtransparent to the light of interest and the subsequent deposition of anabsorbing or phase shifting material into the etched zones. A phaseFresnel zone plate is a staircase approximation to a phase Fresnel lens.The efficiency of the phase Fresnel zone plate increases as the numberof levels is increased. For example, a two-phase Fresnel zone plate canbe shown to have a maximum diffraction efficiency of 40.5%, whereas afour-phase Fresnel zone plate has a maximum diffraction efficiency of81%. Techniques for designing phase Fresnel zone plates with the desiredcharacteristics are known in the art.

Fresnel lenses have been incorporated into advanced optical devicesusing various techniques, for example as imaging optics in illuminationsystems (see, e.g., U.S. Pat. No. 6,002,520), in light emitting devices(see, e.g., U.S. Pat. No. 6,987,613), in solid-state imaging devices(see, e.g, U.S. Pat. No. 7,499,094), in image sensors (see, e.g., U.S.Pat. No. 8,411,375), and in integrated infrared sensors (see, e.g., U.S.Patent Application Publication No. 2013/0043552). The design of theFresnel lenses of the instant disclosure and their integration into theinstant analytical device arrays may be achieved using analogousapproaches.

In highly preferred embodiments, the Fresnel lens of the instantdisclosure is a phase Fresnel zone plate.

Methods for Producing Arrays of Integrated Analytical Devices

In another aspect, the instant disclosure provides methods for producingarrays of integrated analytical devices. As described above, such arraysare useful, for example, in the large-scale sequencing of nucleic acids,such as, for example, genomic sequencing. Such arrays can be produced bya variety of methods. One preferred approach to producing the instantarrays involves the use of microfabrication methods such assemiconductor or MEMS processing methods, which have been highlydeveloped for the production, for example, of integrated circuits.Similar processes have been used to create MEMS (micro electromechanicalsystems) for a variety of applications including inkjet printers,accelerometers, pressure transducers, and displays (such as digitalmicromirror displays (DMDs)). Microfabrication methods can be applied toa large substrate such as a wafer, which can later be diced into manydevices, allowing for the production of many devices at one time.

The methods of the invention may, for example, apply resist processes,such as photoresists, to define structural elements on substrates orother layers. Etching processes may be used to produce three-dimensionalstructures, including component structures in the integrated analyticaldevice. Deposition processes may be used to add layers onto the devices.Other semiconductor processes such as ashing, polishing, release, andliftoff may also be employed to create the structures of the invention,as described in more detail below.

For example, lithographic techniques may be used to define a mask layerout of polymeric materials, such as photoresists, using e.g.,conventional photolithography, e-beam lithography, or the like.Alternatively, lithographic techniques may be applied in conjunctionwith layer deposition methods to deposit metal mask layers, e.g., usingaluminum, gold, platinum, chrome, or other conventionally used metals,or other inorganic mask layers, e.g., silica based substrates such assilicon, SiO₂, or the like. Alternatively, negative tone processes maybe employed to define pillars of resists that correspond to, forexample, apertures. See, e.g., U.S. Pat. No. 7,170,050, which isincorporated by reference herein in its entirety for all purposes. Themask layer may then be deposited over the resist pillars and the pillarsare subsequently removed. In particularly preferred aspects, both theunderlying substrate and the mask layer are fabricated from the samematerial, which in particularly preferred aspects, is a transparentsubstrate material such as an SiO₂-based substrate such as glass,quartz, or fused silica. By providing the mask and underlying layers ofthe same material, one can ensure that the two layers have the sameinteractivity with the environments to which they are exposed, and thusminimize any hybrid surface interactions.

In the case of SiO₂-based substrates and mask layers, conventionalfabrication processes may be employed. In particular, a glass substratebearing the surface exposed waveguide has a layer of resist depositedover its surface. A negative of the mask layer is then defined byappropriate exposure and development of the resist layer to provideresist islands where one wishes to retain access to the underlyingwaveguide. The mask layer is then deposited over the surface and theremaining resist islands are removed, e.g., through a lift off process,to provide the openings to the underlying waveguides. In the case ofmetal layers, deposition may be accomplished through a number of means,including evaporation, sputtering or the like. Such processes aredescribed in, e.g., U.S. Pat. No. 7,170,050. In the case of silica basedmask layers, a chemical vapor deposition (CVD) process may be employedto deposit a silicon layer onto the surface. Following lift off of theresist layer, a thermal oxidation process can convert the mask layer toSiO₂. Alternatively, etching methods may be used to etch access pointsto underlying layers using conventional processes. For example, asilicon layer may be deposited over an underlying substrate. A resistlayer is then deposited over the surface of the silicon layer andexposed and developed to define the pattern of the mask. The accesspoints are then etched from the silicon layer using an appropriatedifferential etch to remove silicon but not the underlying SiO₂substrate. Once the mask layer is defined, the silicon layer is againconverted to SiO₂ using, e.g., a thermal oxidation process.

One aspect of the invention relates to a process for producing arrays ofintegrated analytical devices comprising the steps of: providing asubstrate layer; depositing a filter module layer on the substratelayer; depositing a collection module layer on the filter module layer;patterning and etching the filter module layer and collection modulelayer to form an array of protrusions having tops and sides and havinggaps between the protrusions; depositing a reflective material on thearray of protrusions such that the tops and sides of the protrusionscomprise a reflective layer; depositing a fill material on thereflective layer such that the fill material fills the gaps between theprotrusions; patterning and etching the fill material and reflectivelayer to remove the reflective layer from the tops of the protrusions;depositing a first layer of low n material on the etched fill materialand the tops of the protrusions; depositing a high n material on thefirst layer of low n material; depositing a second layer of low nmaterial on the high n material to form an upper cladding and tocomplete a waveguide module layer disposed on the collection modulelayer; depositing a zero-mode waveguide material on the surface of thewaveguide module layer; and patterning and etching the zero-modewaveguide material to define an array of nanometer-scale aperturespenetrating into the upper cladding of the waveguide module layer.Unless specifically described, the order of the steps of the processesdescribed herein can be altered, where suitable, and, in some cases,steps may be omitted or added.

For example, in some embodiments, the instant fabrication processes mayinclude steps to generate arrays with collection module layers thatcomprise one or more integrated lenses, such as Fresnel lenses. In sucharrays, the integrated lenses may, in certain embodiments, take theplace of the reflective layer, for example the cone-shaped reflectivelayer (1007, 1107, and 1207) of FIGS. 10-12. In some embodiments,however, the instant fabrication processes may be used to generateintegrated analytical devices comprising both one or more integratedlenses and a reflective layer.

One semiconductor fabrication process according an aspect of the instantinvention is shown in FIGS. 18A and 18B, which illustrates thefabrication of an array of integrated analytical devices comprising adielectric filter stack in the cone between the collection layer and thedetection layer. An alternative fabrication process, as illustrated inFIGS. 19A and 19B, results in an array of integrated analytical devicescomprising an absorptive filter stack in the cone between the collectionlayer and the detection layer of the device.

In each of the above exemplary approaches, the process begins with aclean substrate layer. The substrate layer used in the instant methodsof production may be of any suitable rigid material. The substrate layermaterial may comprise, for example, an inorganic oxide material such assilica. A preferred material is a detector layer, such as, for example,a CMOS wafer, i.e., a wafer made up of CMOS sensors or CCD arrays. See,for example, CMOS Imagers From Phototransduction to Image Processing(2004) Yadid-Pecht and Etienne-Cummings, eds.; Springer; CMOS/CCDSensors and Camera Systems (2007) Holst and Lomheim; SPIE Press.

The surface of the substrate may be prepared for deposition by, forexample, a H₂O₂, low HF dip, or another type of oxide-friendly cleaningstep. The second step involves a passivation deposition step, forexample by plasma-enhanced chemical vapor deposition (PECVD) of siliconoxynitride (SiON). This step may also optionally reduce the topographyof the layer. Furthermore, this step may be substituted for differentfilter layers, as shown in more detail in the variant process flow stepsof FIG. 20.

As shown in FIG. 18A, for the fabrication of an array of integratedanalytical devices comprising a dielectric filter stack, the next steppreferably involves deposition of a dielectric filter layer, preferablyby atomic layer deposition. In preferred embodiments, the filter stacksare TiO₂ and SiO₂ or GaP and TiO₂, as described above and illustrated inFIG. 16B. In the alternative process flow of FIGS. 19A and B, for thefabrication of an array of integrated analytical devices comprising anabsorptive filter, the next step involves deposition of an absorptivefilter layer, preferably by spin coat deposition of an epoxy resin, suchas KMPR MicroChem photoresist, or another low autofluorescence resist.In each case, and as described above, the filter layer acts to blocksource light at 532 nm and below.

Following deposition of the filter layer, the remaining steps in theprocess flows of FIGS. 18 and 19, and the variant of FIG. 20, aresimilar. Specifically, a collection module layer is formed on thesurface of the filter module layer, preferably by the deposition ofSiO₂, or another suitable low-loss material, using PECVD techniques. Thestep may, in some embodiments, be omitted, for example if the filterlayer of the device completely fills the reflective cone. The materialmay, in some embodiments, match the material of the bottom cladding inthe waveguide module layer, or it may, in other embodiments, bedifferent.

The patterns of the cones are then defined using an appropriatelithography node, such as I-line or 248 nm node. Following thepatterning step, the cones are etched using an appropriate chemistry(e.g., F chemistry for oxide, ion milling for absorptive filters, etc.).Specific variations in the etching step, such as methods to adjust theslope of the cones and to minimize autofluorescence of the etchedmaterial, may be found, for example, in U.S. Patent ApplicationPublication No. 2010/0099100. In some embodiments of the invention, theorder of the filter deposition and collection cone deposition steps maybe reversed. The system may be optimized to provide better performance,for example by altering the location of the filter in the cone, withinthe scope of the invention, as would be understood by one of ordinaryskill in the art.

Following the cone etching step, a reflective layer is deposited,preferably by sputter deposition. The material used in the reflectivelayer is typically a highly reflective metal, and in preferredembodiments is aluminum, gold, or chromium. In some embodiments,deposition or metallization is accomplished using a conformal depositionprocess, e.g. evaporation.

Following deposition of the reflective layer, the gaps between the conesare filled, preferably using a spin-coat process or PECVD. The materialused to fill the gaps may be chosen depending on the desired function.For example, the material may provide a further absorptive filterfunction, may provide the bottom cladding for the waveguide modulelayer, or may provide a combination of functions. Preferably, the fillmaterial has low autofluorescence and is planarizing. A variety of fillmaterials may be employed for this step, including additional metallayers (or continuous metal layers), inorganic materials, such assilicon, silicon dioxide, polymeric materials, semiconductor materials,or the like. In particularly preferred aspects, a silica based layer isdeposited as the fill layer, and preferably the layer comprises silicondioxide or other glass-like material. Production of a glass fill layermay be accomplished through a number of conventional processes,including the use of spin-on glass materials, such as silsesquioxanes,or through the vapor deposition and subsequent oxidation of silicon filllayers over the substrate.

The surface of the array is next subjected to reverse etch patterning,preferably using an I-line process with a photoresist material. Theresulting reverse mask creates a pattern between the mirrored surfaces,which is subsequently etched using an appropriate chemistry (e.g., Fchemistry for oxide, O₂ for epoxy resin, etc.) as would be understood inthe art. Methods suitable for the etching step may be found, forexample, in U.S. Patent Application Publication No. 2010/0099100.

The etched surface is next subjected to a planarization step toplanarize topography and remove excess oxide from the top of conestructures. The planarization layer may be a hard material such as aspin-on glass, or may be a soft planarization layer. The softplanarization layer may be, for example, a spin-on UV curable organicpolymer such as Summers J91 or SK9. Where the planarization layercomprises a hard material, the planarization is generally polished, forexample with chemical mechanical planarization (CMP). Where theplanarization layer comprises a soft material, such as a UV curepolymer, then after UV cure, oxygen etch may be used to etch away thetop region of the spin-on polymer, analogous to polishing. In someembodiments, the steps of gap fill deposition, reverse etch patterning,reverse pattern etching, and planarization may looped iteratively untilsuitable planarization is achieved.

The planarized surface is next subjected to a pattern definition for topflat mirror etch (TFME) step, preferably using an appropriatelithography node, such as an I-line process, and a photoresist material.The surface is next subjected to a top flat mirror etch (TFME) step,etching a combination of oxide and reflective material, such as Al or Auto uncover the cone layer.

The bottom clad of the waveguide module layer is next deposited,preferably using spin-coat, PECVD, or other suitable methods orcombination of methods. The material deposited is preferably a materialhaving low autofluorescence and low k, such as a low k spin-ondielectric (SOD), SiO2. The deposited surface functions as the claddingfor the waveguide.

The core of the waveguide is then deposited, using plasma-enhancedchemical vapor deposition, atomic layer deposition, or other suitabletechniques or combinations. The core is deposited using a material oflow autofluorescence and high k, such as Si₃N₄, Al₂O₃, or others. Insome embodiments, a waveguide coupler is created on the surface prior todepositing of the core of the waveguide using a pattern/etch loop. Thecoupler and core are deposited using the same material. The pattern forthe waveguide is next defined using an appropriate lithography node,such as I-line or 248 nm node, and a photoresist material. For example,a 300 nm at 5 micro pitch would be a channel waveguide. In someembodiments, dummy waveguides are placed between the functionalwaveguides. In some embodiments, slab waveguides are provided.

The top clad of the waveguide is then deposited by spin-coat, PECVD, orother suitable methods, preferably using a material having lowautofluorescence and low k, such as a low k spin-on dielectric (SOD),SiO2. A confinement material, such as Al or other highly reflective andlow loss material, is next deposited on the surface by, for example,physical vapor deposition (PVD).

The hole pattern for the ZMW nanowells is next defined using anappropriate lithography tool, such as a 193 nm or 248 nm lithographynode, and a photoresist material. The nanowell is aligned as closely aspossible to the underlying cone to ensure efficient transfer of emittedsignals from the ZMW and to minimize cross-talk. The nanowell is nextetched, preferably using a 2-stage process. The nanowell is etched intothe confinement material and the top clad. Suitable materials include,for example, Al, SOG, and SiO₂.

The deep trench of some embodiments of the instant arrays is formed bypattern definition and etching steps using standard methods andmaterials.

After all other process flow steps are complete, the arrays are treatedto remove all residues using a cleaning process step.

As noted above, the detector layer in the integrated analytical devicesof the instant disclosure may include a filter stack to achieve colorseparation, for example as shown in detector layer 612 of FIG. 6 and indetector layer 1205 of FIG. 12. In some embodiments of the instantintegrated analytical devices, color separation may provide furthersignal information about the incorporation step of the sequencingreaction. As shown in FIG. 20, the process flows of FIGS. 18 and 19 maybe modified to allow for the incorporation of color-separation filtersin the detector layer of an integrated analytical device. In particular,the process flow steps shown in FIG. 20 may be used in place of steps1-3 of FIGS. 18 and 19. In other words, a 2-color separation devicewould be generated by following steps 1-8 of FIG. 20 and continuing onwith the collection cone deposition, step 4, of FIGS. 18 and 19.

As mentioned above, the methods of the invention in some cases useresists for defining and producing structures with lithography. Theseresists can be, for example, photoresists or e-beam resists. Thephotoresists can be developed using UV, deep UV, G-line, H-line, I-lineor other suitable wavelength or set of wavelengths. The type of resistthat is used, and therefore the type of instrumentation that is employedfor processing, will depend on the dimensions of the features that arecreated. In many processes described herein, higher resolution resistsand equipment will be used for the production of the aperture whichcorresponds to the reaction volume, where the size of the aperture maybe on the order of 10 nm to 500 nm, and a lower resolution resist andassociated instrumentation is used for the creation of the rest of theintegrated analytical device, which may have features on the dimensionsof 1 micron to 20 microns. Many resists are known in the art, and manyare available commercially from companies such as Rohm and Haas andShipley. The resists used in the processes of the invention may benegative or positive photoresists. Where a process is described hereinusing a negative photoresist, it is to be understood that a suitablepositive photoresist may also be employed where practical, and viceversa. Where appropriate, chemical amplification may also be employed inorder to increase the sensitivity of the resist. The removal of theresist, the cleaning, rinsing, ashing, and drying of the substrate maybe performed as appropriate and as taught and known in the art.

In some cases, the tools used for photolithography of the reactionregion (e.g. ZMW) use photolithography exposure tool capable of creatingstructures having feature sizes of about of 10 nm to about 100 nm. Suchsystems include, for example, an AMSL XT1250 exposure tool.

Etching processes are used in some aspects of the invention in order toproduce the three dimensional features in a substrate or in otherlayers, to fashion, for example, optical elements or lenses, or reactionvolumes such as nanoscale apertures. The etching process that is usedwill depend on the type of material used, the dimensions of thefeatures, and the resist system. In some cases wet etching or wetchemical etching is employed. Electrochemical etching may also beemployed. In some embodiments plasma etching or reactive ion etching(RIE) is used as an etching process. Deep reactive ion etching (DRIE)may also be employed, for example, where structures having high aspectratio are desired. Dry vapor phase etching, for example with xenondifluoride, may also be used. Bulk micromachining or surfacemicromachining may be used as appropriate to create the devicestructures of the disclosure. The etching used in the methods of thedisclosure may be gray-scale etching. The conditions of the resistformation and etching are controlled to produce side walls having thedesired geometries, such as having the desired side-wall angle.

Some processes of the invention involve the deposition of reflectivelayers, or cladding layers. The deposition of these reflective layersmay be accomplished by wet processes including spinning on layers fromsolution, or by gas-phase processes. Suitable processes includeelectroplating, sputter deposition, physical vapor deposition,evaporation, molecular beam epitaxy, atomic layer deposition, andchemical vapor deposition. Metals may be used as the reflective layerand the cladding layer. Suitable metals include gold, nickel, aluminum,chromium, titanium, platinum, and silver. The reflective and/or claddinglayers may comprise aluminum, which may be deposited by sputtering, forexample using a commercially available sputter tool available from CVC,Novellus, or MRC.

Where layers are deposited during the processes of the invention, insome cases, the layers are treated before moving on to the next step inthe process. For example, the deposited layer may be annealed,planarized, cleaned, passivated, or lightly etched in order to improveits properties.

In some methods of the invention, protective layers or sacrificiallayers are deposited. The protective layers may be polymeric layers, ormay be inorganic layers. Suitable protective or sacrificial layersinclude germanium (Ge) and amorphous silicon (a-Si). Protective layersmay be used to produce features as described herein. The type ofmaterial for the protective or sacrificial layer may be chosen for itsselective reactivity, for example to wet chemical etchants. For example,in some cases, the ability to selectively etch germanium with heatedhydrogen peroxide in the presence of silicon dioxide and aluminumresults in its being utilized to produce optical structures combinedwith nanoscale apertures.

In some processes, a pull-back process may be employed. A pull-backprocess generally involves etching in from the edges of a feature withina layer in order to reduce the dimensions of the feature. Pull-back maybe performed using a wet chemical reagent that selectively reacts with alayer which has exposed edges. In some cases a germanium layer is pulledback using hydrogen peroxide.

Some methods employ a polishing step to remove a surface region from asurface. Suitable methods include chemical-mechanical polishing orchemical-mechanical planarization (CMP).

Some methods of the invention incorporate a planarization layer. Themethod for depositing the planarization layer depends on the type ofmaterial that is used. The planarization layer may be a hard material,such as an inorganic material, for example silicon nitride; it may be ametallic material such as aluminum; or it may be a soft material, suchas a polymeric material, e.g. an organic or silicon based polymer. Theplanarization layer may be a glass, such as a silicon dioxide material.In some cases, the planarization layer comprises a spin-on glass such asa silicate, phosphosilicate or siloxane material. Suitable spin-on glassmaterials are available, for example, from Honeywell Corporation. Theplanarization layer may comprise, for example, a glass doped with otheragents to control its melting properties, such a boro-phosphoro-silicateglass (BPSG). Suitable polymeric planarization materials include, forexample, polyimides.

After the arrays of the instant disclosure are complete, such as by, forexample, following the process flows of FIG. 18 or 19, and optionallythe variant steps recited in FIG. 20, the arrays may be furtherprocessed, such as, for example, by separating the arrays intoindividual chips and readying them for sequencing. The furtherprocessing steps will depend on the situation but may typically includethe following treatments: surface treatment (a series of wet/vapor phasetreatments to put down a specific surface that attracts the DNApolymerase enzyme to the bottom of the nanowell); stacking (a process toprotect the top surface of the surface-treated device wafer and, in somecases, creating a well for the sequencing mixture); thinning (a processin which the composite top-plated and surface-treated device wafer maybe thinned—including grinding lapping, polishing, or other treatments);dicing (a process in which the composite wafer is divided intoindividual chips using a standard semiconductor dicing saw); andpackaging (a process involving a standard pick and place tool to mountthe chips onto a substrate and create electrical/optical outputs fordata collection). These further processing steps are either known in theart or are disclosed in references such as U.S. Patent ApplicationPublication Nos. 2008/0176769 and 2011/0183409, which are incorporatedby reference herein in their entireties for all purposes.

As just noted, the arrays of the invention may be incorporated intoanalysis systems for analyzing the multiple reactions occurring in thereaction regions of the array. The arrays described herein typicallyhave reaction regions that are accessible to fluid from the top, andthat are accessible for optical analysis from the bottom. The arrays arethus generally incorporated into a vessel into which a reaction mixtureof interest is introduced. In some cases, the individual reactionregions are all in contact with one volume of fluid, which may have, forexample, multiple nucleic acid template molecules which may be analyzed,and which may have the nucleotides, cofactors, and other additives forcarrying out the reaction to be analyzed.

The vessel that comprises the array may be placed within an instrumentwhich has the appropriate optical components, computer controls, anddata analysis systems. The vessel comprising the array will be heldwithin the instrument such that the reaction conditions, such as thevessel temperature and vessel atmospheric conditions can be controlled.The vessel atmospheric conditions may comprise the makeup of the gasabove the sample, for example the humidity, and the level of othergaseous species such as oxygen.

OTHER ASPECTS

In some aspects, the instant disclosure provides arrays, analyticaldevices, and methods according to the following numbered paragraphs.

1. An array of integrated analytical devices comprising:

a substrate layer;

a filter module layer disposed on the substrate layer;

a collection module layer disposed on or with the filter module layer;

a waveguide module layer disposed on the collection module layer;

a zero-mode waveguide module layer disposed on the waveguide modulelayer;

wherein the zero-mode waveguide module layer comprises at least onenanometer-scale aperture penetrating into the waveguide module layer.

2. The analytical device of paragraph 1, wherein the substrate layer isa detector layer.

3. The analytical device of paragraph 1, wherein the substrate layer isa CMOS wafer.

4. The analytical device of paragraph 1, wherein the filter module layercomprises a dielectric filter.

5. The analytical device of paragraph 1, wherein the filter module layercomprises an absorptive filter.

6. The analytical device of paragraph 1, wherein the detector layercomprises a color-separation layer.

7. A method for producing an array of integrated analytical devicescomprising:

providing a substrate layer;

depositing a filter module layer on the substrate layer;

depositing a collection module layer on the filter module layer;

patterning and etching the filter module layer and the collection modulelayer to form an array of protrusions having tops and sides and havinggaps between the protrusions;

depositing a reflective material on the array of protrusions such thatthe tops and sides of the protrusions comprise a reflective layer;

depositing a fill material on the reflective layer such that the fillmaterial fills the gaps between the protrusions;

patterning and etching the fill material and reflective layer to removethe reflective layer from the tops of the protrusions;

depositing a first layer of low n material on the etched fill materialand the tops of the protrusions;

depositing a high n material on the first layer of low n material;

depositing a second layer of low n material on the high n material toform an upper cladding and to complete a waveguide module layer disposedon the collection module layer;

depositing a zero-mode waveguide material on the surface of thewaveguide module layer;

patterning and etching the zero-mode waveguide material to define anarray of nanometer-scale apertures penetrating into the upper claddingof the waveguide module layer.

8. The method of paragraph 7, further comprising the step of patterningand etching the high n material to define a waveguide.

9. The method of paragraph 7, wherein the substrate layer is a detectorlayer.

10. The method of paragraph 7, wherein the substrate layer is a CMOSwafer.

11. The method of paragraph 7, wherein the filter module layer comprisesa dielectric filter.

12. The method of paragraph 7, wherein the filter module layer comprisesan absorptive filter.

13. The method of paragraph 7, wherein the substrate layer comprises acolor-separation layer.

All patents, patent publications, and other published referencesmentioned herein are hereby incorporated by reference in theirentireties as if each had been individually and specificallyincorporated by reference herein.

While specific examples have been provided, the above description isillustrative and not restrictive. Any one or more of the features of thepreviously described embodiments can be combined in any manner with oneor more features of any other embodiments in the present invention.Furthermore, many variations of the invention will become apparent tothose skilled in the art upon review of the specification. The scope ofthe invention should, therefore, be determined by reference to theappended claims, along with their full scope of equivalents.

What is claimed is:
 1. An array of integrated analytical devicescomprising: a substrate layer; a waveguide module layer disposed on thesubstrate layer; wherein the waveguide module layer comprises a lowerwaveguide cladding material, a waveguide core material, an upperwaveguide cladding material, and an etch hardmask layer disposed betweenthe waveguide core material and the upper waveguide cladding material; azero-mode waveguide module layer disposed on the waveguide module layer;wherein the zero-mode waveguide module layer comprises a plurality ofnanometer-scale apertures penetrating to the waveguide module layer. 2.The array of claim 1, wherein the plurality of nanometer-scale aperturesis formed by etching up to the etch hardmask layer.
 3. The array ofclaim 1, wherein the upper waveguide cladding material is SiO₂.
 4. Thearray of claim 1, wherein the waveguide core material is Si₃N₄.
 5. Thearray of claim 1, wherein the at least one of the plurality ofnanometer-scale apertures is partially backfilled.
 6. The array of claim5, wherein the at least one nanometer-scale aperture is partiallybackfilled using atomic layer deposition or low pressure chemical vapordeposition.
 7. The array of claim 1, wherein the substrate layer is adetector layer.
 8. The array of claim 7, wherein the detector layercomprises a color-separation layer.
 9. The array of claim 1, wherein thesubstrate layer is a CMOS wafer.
 10. The array of claim 1, furthercomprising a collection module layer disposed between the substratelayer and the waveguide module layer.
 11. The array of claim 10, whereinthe collection module layer comprises a Fresnel lens structure.
 12. Thearray of claim 11, wherein the Fresnel lens structure is a phase Fresnelzone plate.
 13. The array of claim 10, further comprising a filtermodule layer disposed between the substrate layer and the collectionmodule layer.
 14. The array of claim 13, wherein the filter module layercomprises a dielectric filter.
 15. The array of claim 13, wherein thefilter module layer comprises an absorptive filter.
 16. The array ofclaim 1, wherein at least one of the plurality of nanometer-scaleapertures comprises a fluid sample comprising a fluorescent species. 17.The array of claim 16, wherein the fluorescent species is afluorescently labeled nucleotide analog.
 18. The array of claim 1,wherein the plurality of nanometer-scale apertures comprise at least 100nanometer-scale apertures.
 19. The array of claim 1, wherein theplurality of nanometer-scale apertures have a density of at least 1000apertures per cm².